Palmitylation is a widespread modification in G-protein-coupled receptors and often a dynamic process. In rhodopsins, palmitylation is static on C322/C323. Red/green (M/LWS) cone opsins have no cysteines at corresponding positions and no palmitylation. Blue (SWS2) cone opsins have a single corresponding cysteine and mass spectrometric analysis showed partial palmitylation of salamander SWS2 cone opsin. Ultraviolet (SWS1) cone opsins have one corresponding cysteine, but only unpalmitylated opsin was observed for mouse and salamander. The results show that the static palmitylation found on rhodopsin is not found on cone opsins and suggest the possibility of an unidentified role for opsin palmitylation in cones.

X-linked cone and cone-rod dystrophies (XLCOD and XLCORD) are a heterogeneous group of progressive disorders that solely or primarily affect cone photoreceptors. Mutations in exon ORF15 of the RPGR gene are the most common underlying cause. In a previous study, we excluded RPGR exon ORF15 in some families with XLCOD. Here, we report genetic mapping of XLCOD to Xq26.1-qter. A significant LOD score was detected with marker DXS8045 (Z(max) = 2.41 [theta = 0.0]). The disease locus encompasses the cone opsin gene array on Xq28. Analysis of the array revealed a missense mutation (c. 529T>C [p. W177R]) in exon 3 of both the long-wavelength-sensitive (LW, red) and medium-wavelength-sensitive (MW, green) cone opsin genes that segregated with disease. Both exon 3 sequences were identical and were derived from the MW gene as a result of gene conversion. The amino acid W177 is highly conserved in visual and nonvisual opsins across species. We show that W177R in MW opsin and the equivalent W161R mutation in rod opsin result in protein misfolding and retention in the endoplasmic reticulum. We also demonstrate that W177R misfolding, unlike the P23H mutation in rod opsin that causes retinitis pigmentosa, is not rescued by treatment with the pharmacological chaperone 9-cis-retinal. Mutations in the LW/MW cone opsin gene array can, therefore, lead to a spectrum of disease, ranging from color blindness to progressive cone dystrophy (XLCOD5).

Cone photoreceptors are responsible for the visual acuity and color vision of the human eye. Red/green cone opsin missense mutations N94K, W177R, P307L, R330Q, and G338E have been identified in subjects with congenital blue cone monochromacy or color-vision deficiency. Studies on disease mechanisms due to these cone opsin mutations have been previously carried out exclusively in vitro, and the reported impairments were not always consistent. Here we expressed these mutants via AAV specifically in vivo in M-opsin knockout mouse cones to investigate their subcellular localization, the pathogenic effects on cone structure, function, and cone viability. We show that these mutations alter the M-opsin structure, function, and localization. N94K and W177R mutants appeared to be misfolded since they localized exclusively in cone inner segments and endoplasmic reticulum. In contrast, P307L, R330Q, and G338E mutants were detected predominately in cone outer segments. Expression of R330Q and G338E, but not P307L opsins, also partially restored expression and correct localization of cone PDE6α' and cone transducin γ and resulted in partial rescue of M-cone-mediated light responses. Expression of W177R and P307L mutants significantly reduced cone viability, whereas N94K, R330Q, and G338E were only modestly toxic. We propose that although the underlying biochemical and cellular defects caused by these mutants are distinct, they all seem to exhibit a dominant phenotype, resembling autosomal dominant retinitis pigmentosa associated with the majority of rhodopsin missense mutations. The understanding of the molecular mechanisms associated with these cone opsin mutants is fundamental to developing targeted therapies for cone dystrophy/dysfunction.

The retinas of mice null for the neural retina leucine zipper transcription factor (Nrl-/-) contain no rods but are populated instead with photoreceptors that on ultrastructural, histochemical, and molecular criteria appear cone like. To characterize these photoreceptors functionally, responses of single photoreceptors of Nrl-/- mice were recorded with suction pipettes at 35-37 degrees C and compared with the responses of rods of WT mice. Recordings were made either in the conventional manner, with the outer segment (OS) drawn into the pipette ("OS in"), or in a novel configuration with a portion of the inner segment drawn in ("OS out"). Nrl-/- photoreceptor responses recorded in the OS-out configuration were much faster than those of WT rods: for dim-flash responses tpeak = 91 ms vs. 215 ms; for saturating flashes, dominant recovery time constants, tau(D) = 110 ms vs. 240 ms, respectively. Nrl-/- photoreceptors in the OS-in configuration had reduced amplification, sensitivity, and slowed recovery kinetics, but the recording configuration had no effect on rod response properties, suggesting Nrl-/- outer segments to be more susceptible to damage. Functional coexpression of two cone pigments in a single mammalian photoreceptor was established for the first time; the responses of every Nrl-/- cell were driven by both the short-wave (S, lambda(max) approximately 360 nm) and the mid-wave (M, lambda(max) approximately 510 nm) mouse cone pigment; the apparent ratio of coexpressed M-pigment varied from 1:1 to 1:3,000 in a manner reflecting a dorso-ventral retinal position gradient. The role of the G-protein receptor kinase Grk1 in cone pigment inactivation was investigated in recordings from Nrl-/-/Grk1-/- photoreceptors. Dim-flash responses of cells driven by either the S- or the M-cone pigment were slowed 2.8-fold and 7.5-fold, respectively, in the absence of Grk1; the inactivation of the M-pigment response was much more seriously retarded. Thus, Grk1 is essential to normal inactivation of both S- and M-mouse cone opsins, but S-opsin has access to a relatively effective, Grk1-independent inactivation pathway.

To convert external light into internal neural signal, vertebrates rely on a special group of proteins, the visual opsins. Four of the five types of visual opsins-short-wavelength sensitive 1 (Sws1), short-wavelength sensitive 2 (Sws2), medium-wavelength sensitive (Rh2), and long-wavelength sensitive (Lws)-are expressed in cone cells for scotopic vision, with the fifth, rhodopsin (Rh1), being expressed in rod cells for photopic vision. Fish often display differing ontogenetic cone opsin expression profiles, which may be related to dietary and/or habitat ontogenetic shift. The western mosquitofish (Gambusia affinis) is an aggressive invader that has successfully colonized every continent except Antarctica. The strong invasiveness of this species may be linked to its visual acuity since it can inhabit turbid waters better than other fishes. By genome screening and transcriptome analysis, we identify seven cone opsin genes in the western mosquitofish, including one sws1, two sws2, one rh2, and three lws. The predicted maximal absorbance wavelength (λmax) values of the respective proteins are 353 nm for Sws1, 449 nm for Sws2a, 408 nm for Sws2b, 516 nm for Rh2-1, 571 nm for Lws-1, and 519 nm for Lws-3. Retention of an intron in the lws-r transcript likely renders this visual opsin gene non-functional. Our real-time quantitative PCR demonstrates that adult male and female western mosquitofish do not differ in their cone opsin expression profiles, but we do reveal an ontogenetic shift in cone opsin expression. Compared to adults, larvae express proportionally more sws1 and less lws-1, suggesting that the western mosquitofish is more sensitive to shorter wavelengths in the larval stage, but becomes more sensitive to longer wavelengths in adulthood.

Three-dimensional retinal organoids can be differentiated from embryonic stem cells/induced pluripotent stem cells (ES/iPS cells) under defined medium conditions. We modified the serum-free floating culture of embryoid body-like aggregates with quick reaggregation (SFEBq) culture procedure to obtain retinal organoids expressing more rod photoreceptors and S- and M-cone opsins.

Independent spatiotemporal control of the stimulation of the five photoreceptor classes requires a display with as many primary lights to probe their isolated spatial and temporal responses. No such system exists with suitable performance properties. We present a system to construct a five-primary display from commercially available three-primary digital light processing projectors. It optimizes the set of five primary lights required to maximize the achievable contrast of a single photoreceptor class in a silent substitution protocol, including where the background chromaticity is first specified. From these chosen five primaries, we describe a method to convert complex three-primary (RGB) images to five-primary representations with complete specification of the photoreceptor excitations at each pixel. Key to enabling this multiple display system with a single HDMI connection is a novel control protocol implemented in a deterministic field-programmable gate array controller that splits the data into five video streams to allow nearly synchronous presentation of primary image data through multiple displays. Each pixel is controlled over 9.5 bits for each primary over a single frame for measurement of threshold-level vision. In addition to a large contrast gamut, the Maxwellian view offers high retinal illumination to support the investigation of five opsin-based responses to complex spatiotemporal images with a truly silent substitution protocol, while avoiding the confounding effects of uncontrolled photoreceptor excitations as occurs in four-primary systems. The customizable primaries facilitate this display translation to species with different photoreceptor spectral responses, and the optics are designed for integration into microscopes for use as a stimulus generator in physiological experiments.

The photosensitive molecule rhodopsin and its relatives consist of a protein moiety - an opsin - and a non-protein moiety - the chromophore retinal. Opsins, which are G-protein-coupled receptors (GPCRs), are found in animals, and more than a thousand have been identified so far. Detailed molecular phylogenetic analyses show that the opsin family is divided into seven subfamilies, which correspond well to functional classifications within the family: the vertebrate visual (transducin-coupled) and non-visual opsin subfamily, the encephalopsin/tmt-opsin subfamily, the Gq-coupled opsin/melanopsin subfamily, the Go-coupled opsin subfamily, the neuropsin subfamily, the peropsin subfamily and the retinal photoisomerase subfamily. The subfamilies diversified before the deuterostomes (including vertebrates) split from the protostomes (most invertebrates), suggesting that a common animal ancestor had multiple opsin genes. Opsins have a seven-transmembrane structure similar to that of other GPCRs, but are distinguished by a lysine residue that is a retinal-binding site in the seventh helix. Accumulated evidence suggests that most opsins act as pigments that activate G proteins in a light-dependent manner in both visual and non-visual systems, whereas a few serve as retinal photoisomerases, generating the chromophore used by other opsins, and some opsins have unknown functions.

Dissecting the genetic basis of phenotypic diversity is one of the fundamental goals in evolutionary biology. Despite growing evidence for gene expression divergence being responsible for the evolution of complex traits, knowledge about the proximate genetic causes underlying these traits is still limited. African cichlids have diverse visual systems, with different species expressing different combinations of seven cone opsin genes. Using opsin expression variation in African cichlids as a model for gene expression evolution, this study aims to investigate the genetic architecture of opsin expression divergence in this group.

Animals have evolved light-sensitive G protein-coupled receptors, known as opsins, to detect coherent and ambient light for visual and nonvisual functions. These opsins have evolved to satisfy the particular lighting niches of the organisms that express them. While many unique patterns of evolution have been identified in mammals for rod and cone opsins, far less is known about the atypical mammalian opsins. Using genomic data from over 400 mammalian species from 22 orders, unique patterns of evolution for each mammalian opsins were identified, including photoisomerases, RGR-opsin (RGR) and peropsin (RRH), as well as atypical opsins, encephalopsin (OPN3), melanopsin (OPN4), and neuropsin (OPN5). The results demonstrate that OPN5 and rhodopsin show extreme conservation across all mammalian lineages. The cone opsins, SWS1 and LWS, and the nonvisual opsins, OPN3 and RRH, demonstrate a moderate degree of sequence conservation relative to other opsins, with some instances of lineage-specific gene loss. Finally, the photoisomerase, RGR, and the best-studied atypical opsin, OPN4, have high sequence diversity within mammals. These conservation patterns are maintained in human populations. Importantly, all mammalian opsins retain key amino acid residues important for conjugation to retinal-based chromophores, permitting light sensitivity. These patterns of evolution are discussed along with known functions of each atypical opsin, such as in circadian or metabolic physiology, to provide insight into the observed patterns of evolutionary constraint.

Color discrimination in the vertebrate retina is mediated by a combination of spectrally distinct cone photoreceptors, each expressing one of multiple cone opsins. The opsin genes diverged early in vertebrate evolution into four classes maximally sensitive to varying wavelengths of light: UV (SWS1), blue (SWS2), green (RH2), and red (LWS) opsins. Although the tetrachromatic cone system is retained in most nonmammalian vertebrate lineages, the transcriptional mechanism underlying gene expression of the cone opsins remains elusive, particularly for SWS2 and RH2 opsins, both of which have been lost in the mammalian lineage. In zebrafish, which have all four cone subtypes, rh2 opsin gene expression depends on a homeobox transcription factor, sine oculis homeobox 7 (Six7). However, the six7 gene is found only in the ray-finned fish lineage, suggesting the existence of another evolutionarily conserved transcriptional factor(s) controlling rh2 opsin expression in vertebrates. Here, we found that the reduced rh2 expression caused by six7 deficiency was rescued by forced expression of six6b, which is a six7-related transcription factor conserved widely among vertebrates. The compensatory role of six6b was reinforced by ChIP-sequencing analysis, which revealed a similar pattern of Six6b- and Six7-binding sites within and near the cone opsin genes. TAL effector nuclease-induced genetic ablation of six6b and six7 revealed that they coordinately regulate SWS2 opsin gene expression. Mutant larvae deficient for these transcription factors showed severely impaired visually driven foraging behavior. These results demonstrate that in zebrafish, six6b and six7 govern expression of the SWS2 and RH2 opsins responsible for middle-wavelength sensitivity, which would be physiologically important for daylight vision.

To analyze the changes of refraction and metabolism of the retinal cones under monochromatic lights in guinea pigs.

Vertebrate behavior is strongly influenced by light. Light receptors, encoded by functional opsin proteins, are present inside the vertebrate brain and peripheral tissues. This expression feature is present from fishes to human and appears to be particularly prominent in diurnal vertebrates. Despite their conserved widespread occurrence, the nonvisual functions of opsins are still largely enigmatic. This is even more apparent when considering the high number of opsins. Teleosts possess around 40 opsin genes, present from young developmental stages to adulthood. Many of these opsins have been shown to function as light receptors. This raises the question of whether this large number might mainly reflect functional redundancy or rather maximally enables teleosts to optimally use the complex light information present under water. We focus on tmt-opsin1b and tmt-opsin2, c-opsins with ancestral-type sequence features, conserved across several vertebrate phyla, expressed with partly similar expression in non-rod, non-cone, non-retinal-ganglion-cell brain tissues and with a similar spectral sensitivity. The characterization of the single mutants revealed age- and light-dependent behavioral changes, as well as an impact on the levels of the preprohormone sst1b and the voltage-gated sodium channel subunit scn12aa. The amount of daytime rest is affected independently of the eyes, pineal organ, and circadian clock in tmt-opsin1b mutants. We further focused on daytime behavior and the molecular changes in tmt-opsin1b/2 double mutants, and found that-despite their similar expression and spectral features-these opsins interact in part nonadditively. Specifically, double mutants complement molecular and behavioral phenotypes observed in single mutants in a partly age-dependent fashion. Our work provides a starting point to disentangle the highly complex interactions of vertebrate nonvisual opsins, suggesting that tmt-opsin-expressing cells together with other visual and nonvisual opsins provide detailed light information to the organism for behavioral fine-tuning. This work also provides a stepping stone to unravel how vertebrate species with conserved opsins, but living in different ecological niches, respond to similar light cues and how human-generated artificial light might impact on behavioral processes in natural environments.

Many animals including birds, reptiles, insects, and teleost fishes can see ultraviolet (UV) light (shorter than 400 nm), which has functional importance for foraging and communication. For coral reef fishes, shallow reef environments transmit a broad spectrum of light, rich in UV, driving the evolution of diverse spectral sensitivities. However, the identities and sites of the specific visual genes that underly vision in reef fishes remain elusive and are useful in determining how evolution has tuned vision to suit life on the reef. We investigated the visual systems of 11 anemonefish (Amphiprioninae) species, specifically probing for the molecular pathways that facilitate UV-sensitivity. Searching the genomes of anemonefishes, we identified a total of eight functional opsin genes from all five vertebrate visual opsin subfamilies. We found rare instances of teleost UV-sensitive SWS1 opsin gene duplications that produced two functionally coding paralogs (SWS1α and SWS1β) and a pseudogene. We also found separate green sensitive RH2A opsin gene duplicates not yet reported in the family Pomacentridae. Transcriptome analysis revealed false clown anemonefish (Amphiprion ocellaris) expressed one rod opsin (RH1) and six cone opsins (SWS1β, SWS2B, RH2B, RH2A-1, RH2A-2, LWS) in the retina. Fluorescent in situ hybridization highlighted the (co-)expression of SWS1β with SWS2B in single cones, and either RH2B, RH2A, or RH2A together with LWS in different members of double cone photoreceptors (two single cones fused together). Our study provides the first in-depth characterization of visual opsin genes found in anemonefishes and provides a useful basis for the further study of UV-vision in reef fishes.

Crypsis increases survival by reducing predator detection. Xenopus laevis tadpoles decode light properties from the substrate to induce two responses: a cryptic coloration response where dorsal skin pigmentation is adjusted to the colour of the substrate (background adaptation) and a behavioural crypsis where organisms move to align with a specific colour surface (background preference). Both processes require organisms to detect reflected light from the substrate. We explored the relationship between background adaptation and preference and the light properties able to trigger both responses. We also analysed which retinal photosensor (type II opsin) is involved. Our results showed that these two processes are segregated mechanistically, as there is no correlation between the preference for a specific background with the level of skin pigmentation, and different dorsal retina-localized type II opsins appear to underlie the two crypsis modes. Indeed, inhibition of melanopsin affects background adaptation but not background preference. Instead, we propose pinopsin is the photosensor involved in background preference. pinopsin mRNA is co-expressed with mRNA for the sws1 cone photopigment in dorsally located photoreceptors. Importantly, the developmental onset of pinopsin expression aligns with the emergence of the preference for a white background, but after the background adaptation phenotype appears. Furthermore, white background preference of tadpoles is associated with increased pinopsin expression, a feature that is lost in premetamorphic froglets along with a preference for a white background. Thus, our data show a mechanistic dissociation between background adaptation and background preference, and we suggest melanopsin and pinopsin, respectively, initiate the two responses.

A nocturnal activity pattern is central to almost all hypotheses on the adaptive origins of primates. This enduring view has been challenged in recent years on the basis of variation in the opsin genes of nocturnal primates. A correspondence between the opsin genes and activity patterns of species in Euarchonta-the superordinal group that includes the orders Primates, Dermoptera (colugos), and Scandentia (treeshrews)-could prove instructive, yet the basic biology of the dermopteran visual system is practically unknown. Here we show that the eye of the Sunda colugo (Galeopterus variegatus) lacks a tapetum lucidum and has an avascular retina, and we report on the expression and spectral sensitivity of cone photopigments. We found that Sunda colugos have intact short wavelength sensitive (S-) and long wavelength sensitive (L-) opsin genes, and that both opsins are expressed in cone photoreceptors of the retina. The inferred peak spectral sensitivities are 451 and 562 nm, respectively. In line with adaptation to nocturnal vision, cone densities are low. Surprisingly, a majority of S-cones coexpress some L-opsin. We also show that the ratio of rates of nonsynonymous to synonymous substitutions of exon 1 of the S-opsin gene is indicative of purifying selection. Taken together, our results suggest that natural selection has favored a functional S-opsin in a nocturnal lineage for at least 45 million years. Accordingly, a nocturnal activity pattern remains the most likely ancestral character state of euprimates.

Cone photoreceptors in fish are typically arranged into a precise, reiterated pattern known as a "cone mosaic." Cone mosaic patterns can vary in different fish species and in response to changes in habitat, yet their function and the mechanisms of their development remain speculative. Zebrafish (Danio rerio) have four cone subtypes arranged into precise rows in the adult retina. Here we describe larval zebrafish cone patterns and investigate a previously unrecognized transition between larval and adult cone mosaic patterns. Cone positions were determined in transgenic zebrafish expressing green fluorescent protein (GFP) in their UV-sensitive cones, by the use of multiplex in situ hybridization labelling of various cone opsins. We developed a "mosaic metric" statistical tool to measure local cone order. We found that ratios of the various cone subtypes in larval and adult zebrafish were statistically different. The cone photoreceptors in larvae form a regular heterotypic mosaic array; i.e., the position of any one cone spectral subtype relative to the other cone subtypes is statistically different from random. However, the cone spectral subtypes in larval zebrafish are not arranged in continuous rows as in the adult. We used cell birth dating to show that the larval cone mosaic pattern remains as a distinct region within the adult retina and does not reorganize into the adult row pattern. In addition, the abundance of cone subtypes relative to other subtypes is different in this larval remnant compared with that of larvae or canonical adult zebrafish retina. These observations provide baseline data for understanding the development of cone mosaics via comparative analysis of larval and adult cone development in a model species.

Many fish alter their expressed visual pigments during development. The number of retinal opsins expressed and their type is normally related to the environment in which they live. Eels are known to change the expression of their rod opsins as they mature, but might they also change the expression of their cone opsins?

Snakes are known to express a rod visual opsin and two cone opsins, only (SWS1, LWS), a reduced palette resulting from their supposedly fossorial origins. Dipsadid snakes in the genus Helicops are highly visual predators that successfully invaded freshwater habitats from ancestral terrestrial-only habitats. Here, we report the first case of multiple SWS1 visual pigments in a vertebrate, simultaneously expressed in different photoreceptors and conferring both UV and violet sensitivity to Helicops snakes. Molecular analysis and in vitro expression confirmed the presence of two functional SWS1 opsins, likely the result of recent gene duplication. Evolutionary analyses indicate that each sws1 variant has undergone different evolutionary paths with strong purifying selection acting on the UV-sensitive copy and dN/dS ∼1 on the violet-sensitive copy. Site-directed mutagenesis points to the functional role of a single amino acid substitution, Phe86Val, in the large spectral shift between UV and violet opsins. In addition, higher densities of photoreceptors and SWS1 cones in the ventral retina suggest improved acuity in the upper visual field possibly correlated with visually guided behaviors. The expanded visual opsin repertoire and specialized retinal architecture are likely to improve photon uptake in underwater and terrestrial environments, and provide the neural substrate for a gain in chromatic discrimination, potentially conferring unique color vision in the UV-violet range. Our findings highlight the innovative solutions undertaken by a highly specialized lineage to tackle the challenges imposed by the invasion of novel photic environments and the extraordinary diversity of evolutionary trajectories taken by visual opsin-based perception in vertebrates.

Cone photopigments (opsins) are crucial elements of, and the first detection module in, color vision. Individual opsins have different wavelength sensitivity patterns, and the temporal and spatial expression patterns of opsins are unique and stringently regulated. Long and middle wavelength-sensitive (L/M) opsins are of the same phylogenetic type. Although the roles of thyroid hormone/TRß2 and COUP-TFs in the transcriptional regulation of L/M opsins have been explored, the detailed mechanisms, including the target sequence in the enhancer of L/M opsins, have not been revealed. We aimed to reveal molecular mechanisms of L/M opsins in vertebrates. Using several human red opsin enhancer/promoter-luciferase reporter constructs, we found that TRß2 increased luciferase activities through the 5'-UTR and intron 3-4 region, whereas the presence of T3 affected only the intron 3-4 region-dependent luciferase activity. Furthermore, COUP-TFII suppressed intron 3-4 region-dependent luciferase activities. However, luciferase expression driven by the mouse M opsin intron 3-4 region was only slightly increased by TRß2, and rather enhanced by COUP-TFII. To determine whether these differential responses reflect differences between primates and rodents, we examined the enhancer/promoter region of the red opsin of the common marmoset. Interestingly, while TRß2 increased 5'-UTR- or intron 3-4 region-driven luciferase expression, as observed for the human red opsin, expression of the latter luciferase was not suppressed by COUP-TFII. In fact, immunostaining of common marmoset retinal sections revealed expression of COUP-TFII and red opsin in the cone cells.